br Materials and methods br Results
Materials and methods
Results RT-PCR showed that both arginase 1 and 2 were expressed in the OB of Korean roe deer (Fig. 1). Immunoblotting detected arginase 1 in the OB of roe deer (Fig. 2A, left lane) at a molecular weight of approximately 37kDa, which is the size of the enzyme in the rat OB (Fig. 2A, right lane). Arginase 2 was also expressed in the OB of roe deer (Fig. 2B, left lane) and the rat (Fig. 2B, right lane) and was approximately 40kDa in size. In the C. pygargus MOB, arginase 1 was detected in the olfactory nerve layer (ONL), glomerulus layer (GL), external plexiform layer (ePL), mitral cell layer (ML), and granule cell layer (GrL) (Fig. 3A). Arginase 1 immunoreactivity was intense in the ML133 HCl of the ONL (Fig. 3D) and the dendrites of the mitral/tufted cells (M/T cells) (Fig. 3E, arrowheads), and it was moderate in the cytoplasm of mitral cells (Fig. 3F, asterisks) and in the GrL (Fig. 3F, arrows). The pattern of arginase 1 expression was similar to that in the rat MOB (Fig. 3B). Arginase 2 was also detected in the ONL, GL, ePL, ML, and GrL (Fig. 4A). Arginase 2 immunoreactivity was moderate in the axons of the ONL (Fig. 4C), the dendrites and cytoplasm (arrowheads) of tufted cells (Fig. 4D), and mitral cells (Fig. 4E, asterisks). Interestingly, arginase 2 immunostained the apical dendrites of mitral cells from the ML to the GL (Fig. 4F, arrows), whereas arginase 2 immunoreactivity was weak in the ePL and GrL (Fig. 4A). In the rat MOB, arginase 2 immunoreactivity showed a similar expression pattern (Fig. 4B). In the C. pygargus AOB, arginase 1 was detected in the GL, the mitral/tufted cell layer (M/TcL), and the GrL (Fig. 5A). Arginase 2 was also detected in all layers (Fig. 5B). In the rat AOB, the expression patterns of arginase 1 (Fig. 5C) and 2 (Fig. 5D) were similar to those of the roe deer. There was a weak positive immunoreaction of both arginase 1 and 2 in the glomerular core of the GL (Figs. 3 D and 4 C) and in negative controls (Fig. 3C), in which primary antibody was omitted. This suggests that both arginase isoforms are expressed in the axons of olfactory sensory neurons and M/T cells. To confirm the cell phenotype of arginase 1- and arginase 2-positive cells, we examined the immunohistochemical staining for GAD, which is a marker for GABAergic interneurons, including periglomerular cells and granule cells (Kim et al., 2015, Kosaka and Kosaka, 2005), and CGRP, which is a marker for M/T cells (Stanic et al., 2010). In the GL, arginase 1-positive cells (arrowheads, Fig. 6A) were also GAD 65/67-positive (arrowheads, Fig. 6B). However, arginase 1-positive mitral cells (arrows, Fig. 6C) were not GAD 65/67-positive (arrows, Fig. 6D) in the ML of mirror sections. Arginase 2-positive mitral cells (asterisks, Fig. 7A) were CGRP-positive (asterisks, Fig. 7B).
Discussion This is the first study to show that both arginase 1 and 2 are immunolocalized in the MOB and AOB of a ruminant, the roe deer, although arginase 1 mRNA was detected in the mouse MOB using in situ hybridization (Yu et al., 2001, Yu et al., 2003) and rat AOB using immunofluorescent staining (Nakamura et al., 1999). We also confirmed that the immunoreactivities of both arginase 1 and 2 in the rat MOB and AOB are similar to that in the roe deer MOB, as in previous in situ hybridization and immunofluorescent studies (Nakamura et al., 1999, Yu et al., 2001, Yu et al., 2003). Regarding the cell arginase phenotype, we found arginase 1 in GAD-positive periglomerular cells in the GL, tufted cells, mitral cells, and some granular cells. This suggests that arginase 1 is involved in both excitatory and inhibitory neurotransmission through its localization in excitatory mitral/tufted cells and inhibitory GAD-positive cells, respectively. The preferential involvement of arginases in either excitatory or inhibitory neurons remains to be studied further. The arginase 2 immunolocalization was similar to that of arginase 1. These findings suggest that the two arginase isoforms are synergistically involved in the catalyzation of l-arginine (Morris, 2009, Wu and Morris, 1998).